Cardiomyocytes in adults have lost the proliferating activity and cardiac transplantation is the only way to treat serious cardiac diseases such as myocardial infarction and cardiomyopathy. In fact, however, owing to a problem of the lack of cardiac tissue donors, there is a pressing need to develop a method of treatment other than cardiac transplantation.
In contrast, use of cardiomyocytes prepared outside the living body to supply with them part of the diseased cardiomyocytes is anticipated to become the most promising way to save patients who have to depend on cardiac transplantation. This approach of treatment is called cell therapy on the heart. To bring this therapy into reality, various trials and errors have been conducted. The methods under review include: using cardiomyocytes or skeletal myoblasts and bone marrow cells or the like that have been extracted from fetuses, neonates or adults; using differentiated embryonic stem cells; and obtaining the stem cells (such as somatic stem cells) which are suggested to exist in the living body has been suggested, and inducing their differentiation (Non-Patent Document 1: Zhonghua Yi Xue Za Zhi 2003, 83, 1818-22).
These methods can be divided into two approaches. One approach involves transplanting cardiomyocytes as cells and in this method, the cardiomyocytes dispersed to single cells are directly injected into a tissue via injection needle (this method is hereinafter referred to as an “injection method”). The other approach involves constructing a tissue or an organ outside the living body (which is hereinafter referred to as a “tissue engineering method”) and this artificial tissue or organ is transferred into the body for treatment.
Various attempts have been made to implement the tissue engineering method and they include: 1) a method in which cardiomyocytes are forms in sheet-like structure, which are then attached onto a tissue (Non-Patent Document 2: Circulation Research 2002, 90(3):e-40); 2) a method in which cardiomyocytes and non-cardiomyocytes are mixed in the same proportions as they are in the cardiac tissue and a three-dimensional structure formed of the mixture is used to replace the tissue; 3) a method in which a three-dimensional structure is formed of the cardiomyocytes dispersed to single cells, with a vascular structure being further constructed, and the three-dimensional structure is substituted for the tissue; and 4) a method in which, rather than replacing the cardiac tissue, a new auxiliary organ that assists in the inherent organ function is transplanted to a site ectopically (Non-Patent Document 3: Circulation Research 2007 2, 100: 263-272).
However, at the present stage where various trials and errors are under way toward clinical therapeutic application, no method has yet exhibited practical data. This is because transplanting cardiomyocytes to the heart involves several problems, such as the inclusion of cells other than cardiomyocytes, low engraftment rate of the transplanted cardiomyocytes, and the inability to exclude components derived from other species.
To use cardiomyocytes as cell masses in transplantation, methods are known that are capable of constructing cell masses including fetal or neonatal rodent cardiomyocytes and, according to a recent report, cell masses were constructed using whole cells (including non-cardiomyocytes) that were derived from the fetal heart (Non-Patent Document 4: Developmental Dynamics 235; 2200-2209, 2006). As regards the transplantation of cardiomyocytes, a case has been reported where fetal mouse cardiomyocytes were transplanted into the hearts of adult mice, which were confirmed to be engrafted (Non-Patent Document Science 1994, 264(5155): 98-101). However, this method of the transplantation of cardiomyocytes involved the use of whole cells to which the whole fetal hearts were dispersed by means of collagenase, so the transplanted cells were composed of a cell population comprising a mixture of cardiomyocytes and non-cardiomyocytes. It is also known that non-purified cardiomyocytes derived from the living body can be transplanted to the heart (Non-Patent Document 5: Science 1994, 264(5155): 98-101; and Non-Patent Document 1: Zhonghua Yi Xue Za Zhi 2003, 83, 1818-22).
Also known is a method in which, in the process of differentiation of embryoid bodies from ES cells, the embryoid bodies are incompletely treated with a proteolytic enzyme, whereupon a population comprising cell masses that are rich in cardiomyocytes and those which are not is obtained and then is subjected to density gradient centrifugation, thereby obtaining cell masses that contain up to about 70% of cardiomyocytes (Patent Document 1: US 2005-0214938 A).
However, each of those methods involves the use of a cell population that also contains cells other than cardiomyocytes and contamination of such cells other than cardiomyocytes may have the potential to cause serious unpredictable side effects that may threaten the life of a patient after transplantation. Under the circumstances, it is considered necessary that cardiomyocytes to be subjected to transplantation therapy should be used after purification.
Several reports have described achievements in transplanting unpurified, ES cell-derived cardiomyocytes to the heart and allowing them to be engrafted thereafter (Non-Patent Document 6: Cardiovasc Res. 2007 May 17; Non-Patent Document 7: Stem Cells. 2007 May 31; and Non-Patent Document 8: FASEB J. 2007 Apr. 13). According to a recent paper, however, which discussed purifying ES cell-derived cardiomyocytes and injecting them into the heart, the engraftment rate of the transplanted cardiomyocytes was extremely low and no cardiomyocytes were found to be engrafted (i.e., those survived within the host organ and remained adherent in it for an extended period of time); as it turned out, the purified, ES cell-derived cardiomyocytes were not able to be engrafted after they were transplanted into an individual (the living body) (Non-Patent Document 9: J Exp Med. 2006; 203:2315-27.)
This report has brought light to the difficulty in causing purified cardiomyocytes to be engrafted after transplantation. In order to solve this problem, a method was discovered in the same report that involved transplanting the ES cell-derived cardiomyocytes in admixture with mouse embryonic fibroblasts with a for the purpose of enhancing their engraftment rate after transplantation (Non-Patent Document 9: J Exp Med. 2006 Oct. 2; 203(10): 2315-27). This shows that no known methods are capable of transplanting purified ES cell-derived cardiomyocytes to remain engrafted while retaining their purity.
In addition, in order to prepare cell transplants that are intended for use in therapy on the human body, serum and other factors that are derived from other animals must be excluded. In the method of preparing cardiomyocytes to be used in transplantation, culture is usually performed in the presence of serum; but it is known that under serum-free conditions, human ES cells can form embryoid bodies, which contain cardiomyocytes in comparable amounts to those obtained by the usual culture in the presence of serum (Non-Patent Document 10: Stem cells and development 15:931-941, 2006). However, no known reports including this report have described a case of transplanting cardiomyocytes that were prepared without using factors such as serum that were derived from other animals.
Thus, in order that the cardiomyocytes could be successfully transplanted to the heart, several problems, such as the inclusion of cells other than cardiomyocytes, the low engraftment rate of transplanted cardiomyocytes and the inability to exclude components derived from other species, must be solved altogether.
Further, in connection with their transplantation to the cardiac tissue, it is contemplated to transplant cardiomyocytes in the form of so-called “cell sheets”. As regards the preparation of cell sheets, it is known that neonatal cardiomyocytes are used to form a singlelayered sheet and up to three of such sheets can be stratified in vitro (Non-Patent Document 11: FASEB J. 2006 April; 20(6): 708-10). However, this document also states that, on account of limited oxygen permeability, the cell sheets cannot be made any thicker without neovascularization to the cell sheet, and it is not possible yet to prepare a desired cell sheet that fits the size of the diseased tissue of the heart.
As described above, the stated of the art is such that the preparation of cardiomyocytes to be used in transplantation and the transplantation of those cardiomyocytes need further improvements from the viewpoint of practical feasibility.
Patent Document 1: US 2005-0214938 A
Non-Patent Document 1: Zhonghua Yi Xue Za Zhi 2003, 83, 1818-22
Non-Patent Document 2: Circulation Research 2002, 90(3):e-40
Non-Patent Document 3: Circulation Research 2007 2, 100: 263-272
Non-Patent Document 4: Developmental Dynamics 235; 2200-2209, 2006
Non-Patent Document 5: Science 1994, 264(5155): 98-101
Non-Patent Document 6: Cardiovasc Res. 2007 May 17 (Flk1(+) cardiac stem/progenitor cells derived from embryonic stem cells improve cardiac function in a dilated cardiomyopathy mouse model)
Non-Patent Document 7: Stem Cells. 2007 May 31 (Differentiation in vivo of Cardiac Committed Human Embryonic Stem Cells in Post-Myocardial Infarcted Rats)
Non-Patent Document 8: FASEB J. 2007 Apr. 13 (Identification and selection of cardiomyocytes during human embryonic stem cell differentiation)
Non-Patent Document 9: J Exp Med. 2006 Oct. 2; 203(10): 2315-27
Non-Patent Document 10: Stem Cell and Development 15: 931-941, 2006
Non-Patent Document 11: FASEB J. 2006 April; 20(6): 708-10